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        FOOTPRINTING WITH DNASE1

        互联网

        1565

        John Mundy ,Institute of Molecular Biology,Copenhagen,Denmark

        http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#Footprint

        1)probe: same as used for gelshift),isolated by isotacelectrophoresis w/out EtOH ppt which can enature dsDNA

        2)probe mix/rxn: volumes x # samples

        1μl probe (0.1©0.5ng or 10©20kcpm)

        0.15μl 20mM EDTA

        0.4μl 10μg/μl dIdC or dAdT (from gel shift assay)

        0.5μl H2O

        3)DNA se mix: made up near end of binding incubations.DNA se l(Worthington DPFF,Cat

        #LS0006330,lot #58A047,5mg)is 1mg/ml in150mM NaCl,50% glycerol,store at ©20℃.

        Try 3 different [s] ofDNA se mix (A,B,C)

        1,2 & 3μl stock DNA se1

        2μl 1M MgCl2

        ©> 100μl H2O

        4)binding rxn: components titrated & optimized by gel shiftassays

        2μl probe mix

        Xul extract

        ©>18μl NEB (see nucprp.ptc)

        30' RT

        5)DNA se rxn: add 2μl DNA se mix to binding rxn

        inc 1' RT

        stop w/ 100μl DNA se stop mix:

        stock/50ml

        6M Urea 18g

        0.4M NaCl 6.6ml 3M

        1% SDS 5ml 10%

        20mM EDTA 4ml 250mM

        10mM Tris 8 0.5ml 1M

        0.8M NH4OAc 5ml 8M

        10μg/ml glycogen 50μl 10mg/ml

        5)P/CHCl3 ext

        6)EtOH ppt

        7)PAGE: Resuspend carefully in 8μl sequencing sample buffer (5'vortex,5' 60℃,1'vortex,2' 90℃,spin,transfer to new tube,count cpm).Load equal counts on 6% or gradient sequencing gel.

        Notes: If extract inhibits DNA se,add 0.1©0.3μl extra DNA se mixto binding rxns.

        DNA se requires Mg,some factors are inactivatedby it! Remember μg/KB x 0.66 = picomole thus 1ng of 300bp probe =2 femptomole.

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